888 Citations
Prostanoids are a series of bioactive lipid metabolites that function in an autacoid manner via activation of cognate G-protein-coupled receptors GPCRs Here we report the crystal structure of human prostaglandin PG E receptor subtype EP bound to endogenous ligand PGE at resolution The structure reveals important insights into the activation mechanism of prostanoid receptors and provides a molecular basis for the binding modes of endogenous ligands
Misoprostol is a life-saving drug in many developing countries for women at risk of post-partum hemorrhaging due to its affordability stability ease of administration and clinical efficacy However misoprostol lacks receptor and tissue selectivities and thus its use is accompanied by a number of serious side-effects The development of pharmacological agents combining the advantages of misoprostol with improved selectivity is hindered by the absence of atomic details of misoprostol action in labor induction Here we present the resolution crystal structure of misoprostol free-acid form bound to the myometrium labor-inducing prostaglandin E receptor EP The active-state structure reveals a completely enclosed ... More
Misoprostol is a life-saving drug in many developing countries for women at risk of post-partum hemorrhaging due to its affordability, stability, ease of administration and clinical efficacy. However, misoprostol lacks receptor and tissue selectivities and thus its use is accompanied by a number of serious side-effects. The development of pharmacological agents combining the advantages of misoprostol with improved selectivity is hindered by the absence of atomic details of misoprostol action in labor induction. Here, we present the 2.5 Å resolution crystal structure of misoprostol free-acid form bound to the myometrium labor-inducing prostaglandin E2 receptor 3 (EP3). The active-state structure reveals a completely enclosed binding pocket containing a structured water molecule that coordinates misoprostol ring structure. Modelling of selective agonists in EP3 structure reveals rationales for selectivity. These findings will provide the basis for the next generation of uterotonic drugs that will be suitable for administration in low resource settings. Less
Prostaglandin E receptor EP a G-protein-coupled receptor is involved in disorders such as cancer and autoimmune disease Here we report the crystal structure of human EP in complex with its antagonist ONO-AE - and an inhibitory antibody at resolution The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer proximal to the highly conserved Arg residue in the seventh transmembrane domain Functional and docking studies demonstrate that the natural agonist PGE binds in a similar manner This structural information also provides insight into the ligand ... More
Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family. Less
Toxin antitoxin TA systems are involved in diverse physiological processes in prokaryotes but their exact role in Mycobacterium tuberculosis Mtb virulence and in vivo stress adaptation has not been extensively studied Here we demonstrate that the VapBC TA module is essential for Mtb to establish infection in guinea pigs RNA-sequencing revealed that overexpression of VapC toxin results in metabolic slowdown suggesting that modulation of the growth rate is an essential strategy for in vivo survival Interestingly overexpression of VapC resulted in the upregulation of chromosomal TA genes suggesting the existence of highly coordinated crosstalk among TA systems In this study ... More
Toxin–antitoxin (TA) systems are involved in diverse physiological processes in prokaryotes, but their exact role in Mycobacterium tuberculosis (Mtb) virulence and in vivo stress adaptation has not been extensively studied. Here, we demonstrate that the VapBC11 TA module is essential for Mtb to establish infection in guinea pigs. RNA-sequencing revealed that overexpression of VapC11 toxin results in metabolic slowdown, suggesting that modulation of the growth rate is an essential strategy for in vivo survival. Interestingly, overexpression of VapC11 resulted in the upregulation of chromosomal TA genes, suggesting the existence of highly coordinated crosstalk among TA systems. In this study, we also present the crystal structure of the VapBC11 heterooctameric complex at 1.67 Å resolution. Binding kinetic studies suggest that the binding affinities of toxin–substrate and toxin–antitoxin interactions are comparable. We used a combination of structural studies, molecular docking, mutational analysis and in vitro ribonuclease assays to enhance our understanding of the mode of substrate recognition by the VapC11 toxin. Furthermore, we have also designed peptide-based inhibitors to target VapC11 ribonuclease activity. Taken together, we propose that the structure-guided design of inhibitors against in vivo essential ribonucleases might be a novel strategy to hasten clearance of intracellular Mtb. Less
URI http hdl handle net Content Type Thesis Files Elion-Jourard Shira S MSc thesis pdf Permanent link https hdl handle net
Metallo- -Lactamases MBLs protect bacteria from almost all -lactam antibiotics Verona integron-encoded MBL VIM enzymes are among the most clinically important MBLs with VIM- increasing in carbapenem-resistant Enterobacteriaceae Escherichia coli Klebsiella pneumoniae that are among the hardest bacterial pathogens to treat VIM enzymes display sequence variation at residues and that in related MBLs are conserved and participate in substrate binding How they accommodate this variability while retaining catalytic efficiency against a broad substrate range has remained unclear Here we present crystal structures of VIM- and its complexes with a substrate-mimicking thioenolate inhibitor ML F that restores meropenem activity against a ... More
Metallo-β-Lactamases (MBLs) protect bacteria from almost all β-lactam antibiotics. Verona integron-encoded MBL (VIM) enzymes are among the most clinically important MBLs, with VIM-1 increasing in carbapenem-resistant Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) that are among the hardest bacterial pathogens to treat. VIM enzymes display sequence variation at residues (224 and 228) that in related MBLs are conserved and participate in substrate binding. How they accommodate this variability, while retaining catalytic efficiency against a broad substrate range, has remained unclear. Here, we present crystal structures of VIM-1 and its complexes with a substrate-mimicking thioenolate inhibitor, ML302F, that restores meropenem activity against a range of VIM-1 producing clinical strains, and the hydrolysed product of the carbapenem meropenem. Comparison of these two structures identifies a water-mediated hydrogen bond, between the carboxylate group of substrate/inhibitor and the backbone carbonyl of the active site zinc ligand Cys221, that is common to both complexes. Structural comparisons show that the responsible Cys221-bound water is observed in all known VIM structures, participates in carboxylate binding with other inhibitor classes, and thus effectively replicates the role of the conserved Lys224 in analogous complexes with other MBLs. These results provide a mechanism for substrate binding that permits the variation at positions 224 and 228 that is a hallmark of VIM MBLs. Less
Human respiratory syncytial virus HRSV is a negative-stranded RNA virus that causes a globally prevalent respiratory infection which can cause lifethreatening illness particularly in the young elderly and immunocompromised HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase RdRp For replication this complex comprises the phosphoprotein P and the large protein L whereas for transcription the M - protein is also required M - is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M - surface such that binding of these ... More
Human respiratory syncytial virus (HRSV) is a negative-stranded RNA virus that causes a globally prevalent respiratory infection, which can cause lifethreatening illness, particularly in the young, elderly, and immunocompromised. HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase (RdRp). For replication, this complex comprises the phosphoprotein (P) and the large protein (L), whereas for transcription, the M2-1 protein is also required. M2-1 is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M2-1 surface, such that binding of these partners is mutually exclusive. The molecular basis for the transcriptional requirement of M2-1 is unclear, as is the consequence of competition between P and RNA for M2-1 binding, which is likely a critical step in the transcription mechanism. Here, we report the crystal structure at 2.4 Å of M2-1 bound to the P interaction domain, which comprises P residues 90 to 110. The P90 – 110 peptide is alpha helical, and its position on the surface of M2-1 defines the orientation of the three transcriptase components within the complex. The M2-1/P interface includes ionic, hydrophobic, and hydrogen bond interactions, and the critical contribution of these contacts to complex formation was assessed using a minigenome assay. The affinity of M2-1 for RNA and P ligands was quantified using fluorescence anisotropy, which showed high-affinity RNAs could outcompete P. This has important implications for the mechanism of transcription, particularly the events surrounding transcription termination and synthesis of poly(A) sequences. Less
Human muscarinic receptor M is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors Muscarinic receptors are targets for multiple neurodegenerative diseases The challenge has been designing subtype selective ligands against one of the five muscarinic receptors We report high resolution structures of a thermostabilized mutant M receptor bound to a subtype selective antagonist AF-DX and a non-selective antagonist NMS The thermostabilizing mutation S R in M was predicted using a theoretical strategy previously developed in our group Comparison of the crystal structures and pharmacological properties of the M receptor shows that the ... More
Human muscarinic receptor, M2 is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype selective ligands against one of the five muscarinic receptors. We report high resolution structures of a thermostabilized mutant M2 receptor bound to a subtype selective antagonist AF-DX 384 and a non-selective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, that is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular Dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptor compared to M3 receptor leads to subtype selectivity of AF-DX 384. Less
Kinesin- transports numerous cellular cargoes along microtubules The kinesin- light chain KLC mediates cargo binding and regulates kinesin- motility To investigate the molecular basis for kinesin- recruitment and activation by cargoes we solved the crystal structure of the KLC tetratricopeptide repeat TPR domain bound to the cargo JIP This combined with biophysical and molecular evolutionary analyses reveals a kinesin- cargo binding site located on KLC TPR which is conserved in homologs from sponges to humans In the complex JIP crosslinks two KLC TPR domains via their TPR s We show that TPR forms a dimer interface that mimics JIP binding ... More
Kinesin-1 transports numerous cellular cargoes along microtubules. The kinesin-1 light chain (KLC) mediates cargo binding and regulates kinesin-1 motility. To investigate the molecular basis for kinesin-1 recruitment and activation by cargoes, we solved the crystal structure of the KLC2 tetratricopeptide repeat (TPR) domain bound to the cargo JIP3. This, combined with biophysical and molecular evolutionary analyses, reveals a kinesin-1 cargo binding site, located on KLC TPR1, which is conserved in homologs from sponges to humans. In the complex, JIP3 crosslinks two KLC2 TPR domains via their TPR1s. We show that TPR1 forms a dimer interface that mimics JIP3 binding in all crystal structures of the unbound KLC TPR domain. We propose that cargo-induced dimerization of the KLC TPR domains via TPR1 is a general mechanism for activating kinesin-1. We relate this to activation by tryptophan-acidic cargoes, explaining how different cargoes activate kinesin-1 through related molecular mechanisms. Less
Chagas disease caused by Trypanosoma cruzi affects millions of people in South America and no satisfactory therapy exists especially for its life threatening chronic phase We targeted the Proline Racemase of T cruzi which is present in all stages of the parasite life cycle to discover new inhibitors against this disease The first published crystal structures of the enzyme revealed that the catalytic site is too small to allow any relevant drug design In previous work to break through the chemical space afforded to virtual screening and drug design we generated intermediate models between the open ligand free and closed ... More
Chagas disease, caused by Trypanosoma cruzi, affects millions of people in South America and no satisfactory therapy exists, especially for its life threatening chronic phase. We targeted the Proline Racemase of T. cruzi, which is present in all stages of the parasite life cycle, to discover new inhibitors against this disease. The first published crystal structures of the enzyme revealed that the catalytic site is too small to allow any relevant drug design. In previous work, to break through the chemical space afforded to virtual screening and drug design, we generated intermediate models between the open (ligand free) and closed (ligand bound) forms of the enzyme. In the present work, we co-crystallized the enzyme with the selected inhibitors and found that they were covalently bound to the catalytic cysteine residues in the active site, thus explaining why these compounds act as irreversible inhibitors. These results led us to the design of a novel, more potent specific inhibitor, NG-P27. Co-crystallization of this new inhibitor with the enzyme allowed us to confirm the predicted protein functional motions and further characterize the chemical mechanism. Hence, the catalytic Cys300 sulfur atom of the enzyme attacks the C2 carbon of the inhibitor in a coupled, regiospecific—stereospecific Michael reaction with trans-addition of a proton on the C3 carbon. Strikingly, the six different conformations of the catalytic site in the crystal structures reported in this work had key similarities to our intermediate models previously generated by inference of the protein functional motions. These crystal structures span a conformational interval covering roughly the first quarter of the opening mechanism, demonstrating the relevance of modeling approaches to break through chemical space in drug design. Less
The XChem facility at Diamond Light Source offers fragment screening by X-ray crystallography as a general access user program The main advantage of X-ray crystallography as a primary fragment screen is that it yields directly the location and pose of the fragment hits whether within pockets of interest or merely on surface sites this is the key information for structure-based design and for enabling synthesis of follow-up molecules Extensive streamlining of the screening experiment at XChem has engendered a very active user program that is generating large amounts of data in academic and industry groups generated datasets of uniquely soaked ... More
The XChem facility at Diamond Light Source offers fragment screening by X-ray crystallography as a general access user program. The main advantage of X-ray crystallography as a primary fragment screen is that it yields directly the location and pose of the fragment hits, whether within pockets of interest or merely on surface sites: this is the key information for structure-based design and for enabling synthesis of follow-up molecules. Extensive streamlining of the screening experiment at XChem has engendered a very active user program that is generating large amounts of data: in 2017, 36 academic and industry groups generated 35,000 datasets of uniquely soaked crystals. It has also generated a large number of learnings concerning the main remaining bottleneck, namely, obtaining a suitable crystal system that will support a successful fragment screen. Here we discuss the practicalities of generating screen-ready crystals that have useful electron density maps, and how to ensure they will be successfully reproduced and usable at a facility outside the home lab. Less
Inteins remove themselves from a precursor protein by protein splicing Due to the concomitant structural changes of the host protein this self-processing reaction has enabled many applications in protein biotechnology and chemical biology We show that the evolved M mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking extein position compared to the parent intein in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein Surprisingly side chains with increased steric bulk compared to the native Gly residue including D-amino acids were found to compensate for ... More
Inteins remove themselves from a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking (−1) extein position compared to the parent intein, in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein. Surprisingly, side chains with increased steric bulk compared to the native Gly(−1) residue, including D-amino acids, were found to compensate for the essential block B histidine in His73Ala mutants in the initial N–S acyl shift of the protein splicing pathway. In the case of the M86 intein, large (−1) side chains can even rescue protein splicing activity as a whole. With the comparison of three crystal structures, namely of the M86 intein as well as of its Gly(−1)Phe and Gly(−1)Phe/His73Ala mutants, our data supports a model in which the intein's active site can exert a strain by varying mechanisms on the different angles of the scissile bond at the extein–intein junction to effect a ground-state destabilization. The compensatory mechanism of the block B histidine is the first example for the direct functional role of an extein residue in protein splicing. It sheds new light on the extein–intein interplay and on possible consequences of their co-evolution as well as on the laboratory engineering of improved inteins. Less
A statistical model enables auto-calibration of second harmonic generation SHG images for quantifying trace crystallinity within amorphous solid dispersions ASDs over a wide dynamic range of crystallinity In this paper we demonstrate particle-counting approaches for quantifying trace crystallinity combined with analytical expressions correcting for particle overlap bias in higher crystallinity regimes to extend the continuous dynamic range of standard particle-counting algorithms through to the signal averaging regime The reliability of the values recovered by these expressions was demonstrated with simulated data as well as experimental data obtained for an amorphous solid dispersion formulation containing evacetrapib an Eli Lilly and Company ... More
A statistical model enables auto-calibration of second harmonic generation (SHG) images for quantifying trace crystallinity within amorphous solid dispersions (ASDs) over a wide dynamic range of crystallinity. In this paper, we demonstrate particle-counting approaches for quantifying trace crystallinity, combined with analytical expressions correcting for particle overlap bias in higher crystallinity regimes to extend the continuous dynamic range of standard particle-counting algorithms through to the signal averaging regime. The reliability of the values recovered by these expressions was demonstrated with simulated data as well as experimental data obtained for an amorphous solid dispersion formulation containing evacetrapib, an Eli Lilly and Company compound. Since particle counting independently recovers the crystalline volume and the SHG intensity, the average SHG intensity per unit volume can be used as an internal calibrant for quantifying crystallinity at higher volume fractions, for which particle counting is no longer applicable. Less
The closely related type III secretion system zinc metalloprotease effector proteins GtgA GogA and PipA are translocated into host cells during Salmonella infection They then cleave nuclear factor -light-chain-enhancer of activated B cells NF- B transcription factor subunits dampening activation of the NF- B signaling pathway and thereby suppressing host immune responses We demonstrate here that GtgA GogA and PipA cleave a subset of NF- B subunits including p RelB and cRel but not NF- B and NF- B whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF- B ... More
The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during Salmonella infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel but not NF-κB1 and NF-κB2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF-κB subunits. Mutational analysis of NF-κB subunits revealed that a single nonconserved residue in NF-κB1 and NF-κB2 that corresponds to the P1′ residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1′ residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-κB subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-κB activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence. Less
Purine nucleoside phosphorylases PNPs play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway Here we present the structural and kinetic characterization of a new PNP isoform from S mansoni named as SmPNP Screening of different ligands using a thermofluorescence approach indicated cytidine and cytosine as potential ligands The binding of cytosine was confirmed by isothermal titration calorimetry with a KD of M and kinetic parameters for cytidine catalysis were obtained by ITC resulting in a KM of M SmPNP also displays catalytic activity against inosine and adenosine making it ... More
Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, named as SmPNP2. Screening of different ligands using a thermofluorescence approach indicated cytidine and cytosine as potential ligands. The binding of cytosine was confirmed by isothermal titration calorimetry, with a KD of 27 μM, and kinetic parameters for cytidine catalysis were obtained by ITC resulting in a KM of 76.3 μM. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystallographic structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capability of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site allowing an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site deepens the understanding of the correlation between structure and nucleotide selectivity, offering new ways to analyses PNP activity. Less
Neisserial heparin binding antigen NHBA is one of three main recombinant protein antigens in CMenB a vaccine for the prevention of invasive meningococcal disease caused by Neisseria meningitidis serogroup B NHBA is a surface-exposed lipoprotein composed of a predicted disordered N-terminal region an arginine-rich region that binds heparin and a C-terminal domain that folds as an anti-parallel -barrel and that upon release after cleavage by human proteases alters endothelial permeability NHBA induces bactericidal antibodies in humans and NHBA-specific antibodies elicited by the CMenB vaccine contribute to serum bactericidal activity the correlate of protection To better understand the structural bases of ... More
Neisserial heparin binding antigen (NHBA) is one of three main recombinant protein antigens in 4CMenB, a vaccine for the prevention of invasive meningococcal disease caused by Neisseria meningitidis serogroup B. NHBA is a surface-exposed lipoprotein composed of a predicted disordered N-terminal region, an arginine-rich region that binds heparin, and a C-terminal domain that folds as an anti-parallel β-barrel and that upon release after cleavage by human proteases alters endothelial permeability. NHBA induces bactericidal antibodies in humans, and NHBA-specific antibodies elicited by the 4CMenB vaccine contribute to serum bactericidal activity, the correlate of protection. To better understand the structural bases of the human antibody response to 4CMenB vaccination and to inform antigen design, we used X-ray crystallography to elucidate the structures of two C-terminal fragments of NHBA, either alone or in complex with the Fab derived from the vaccine-elicited human monoclonal antibody 5H2, and the structure of the unbound Fab 5H2. The structures reveal details on the interaction between an N-terminal β-hairpin fragment and the β-barrel, and explain how NHBA is capable of generating cross-reactive antibodies through an extensive conserved conformational epitope that covers the entire C-terminal face of the β-barrel. By providing new structural information on a vaccine antigen and on the human immune response to vaccination, these results deepen our molecular understanding of 4CMenB, and might also aid future vaccine design projects. Less
Frizzled receptors FZDs are class-F G-protein-coupled receptors GPCRs that function in Wnt signalling and are essential for developing and adult organisms As central mediators in this complex signalling pathway FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research Here we present an atomic-resolution structure of the human Frizzled receptor FZD transmembrane domain in the absence of a bound ligand The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed and is distinct from all other GPCR structures reported so far Within this ... More
Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs. Less
The avian influenza A H N virus continues to cause human infections in China and is a major ongoing public health concern Five epidemic waves of A H N infection have occurred since and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves Moreover highly pathogenic avian influenza HPAI A H N viruses were also isolated during the fifth epidemic wave Here we present a detailed structural and biochemical analysis of the surface hemagglutinin HA antigen ... More
The avian influenza A(H7N9) virus continues to cause human infections in China and is a major ongoing public health concern. Five epidemic waves of A(H7N9) infection have occurred since 2013, and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves. Moreover, highly pathogenic avian influenza (HPAI) A(H7N9) viruses were also isolated during the fifth epidemic wave. Here, we present a detailed structural and biochemical analysis of the surface hemagglutinin (HA) antigen from viruses isolated during this recent epidemic wave. Results highlight that, compared to the 2013 virus HAs, the fifth-wave virus HAs remained a weak binder to human glycan receptor analogs. We also studied three mutations, V177K-K184T-G219S, that were recently reported to switch a 2013 A(H7N9) HA to human-type receptor specificity. Our results indicate that these mutations could also switch the H7 HA receptor preference to a predominantly human binding specificity for both fifth-wave H7 HAs analyzed in this study. Less
Whirlin is a protein essential to sensory neurons Its defects are responsible for nonsyndromic deafness or for the Usher syndrome a condition associating congenital deafness and progressive blindness This large multidomain scaffolding protein is expressed in three isoforms with different functions and localizations in stereocilia bundles of hearing hair cells or in the connecting cilia of photoreceptor cells The HHD domain of whirlin is the only domain shared by all isoforms but its function remains unknown In this article we report its crystal structure in two distinct conformations a monomeric five-helix bundle similar to the known structure of other HHD ... More
Whirlin is a protein essential to sensory neurons. Its defects are responsible for nonsyndromic deafness or for the Usher syndrome, a condition associating congenital deafness and progressive blindness. This large multidomain scaffolding protein is expressed in three isoforms with different functions and localizations in stereocilia bundles of hearing hair cells or in the connecting cilia of photoreceptor cells. The HHD2 domain of whirlin is the only domain shared by all isoforms, but its function remains unknown. In this article, we report its crystal structure in two distinct conformations, a monomeric five-helix bundle, similar to the known structure of other HHD domains, and a three-helix bundle organized as a swapped dimer. Most of the hydrophobic contacts and electrostatic interactions that maintain the globular monomeric form are conserved at the protomer interface of the dimer. NMR experiments revealed that the five-helix conformation is predominant in solution, but exhibits increased dynamics on one face encompassing the hinge loops. Using NMR and SAXS, we also show that HHD2 does not interact with its preceding domains. Our findings suggest that structural plasticity might play a role in the function of the HHD2 domain. Less
Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers FELs for serial femtosecond crystallography measurements The high X-ray intensity in the focus initiates an explosion of the microjet and sample With the advent of X-ray FELs with megahertz rates the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using nm radiation The operation of gas-dynamic nozzles that ... More
Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3 nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80 m s-1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5 MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments. Less
Rotaviruses RVs cause life-threatening diarrhea in infants and children worldwide Recent biochemical and epidemiological studies underscore the importance of histo-blood group antigens HBGA as both cell attachment and susceptibility factors for the globally dominant P P and P genotypes of human RVs How these genotypes interact with HBGA is not known Here our crystal structures of P and a neonate-specific P VP s alone and in complex with H-type I HBGA reveal a unique glycan binding site that is conserved in the globally dominant genotypes and allows for the binding of ABH HBGAs consistent with their prevalence Remarkably the VP ... More
Rotaviruses (RVs) cause life-threatening diarrhea in infants and children worldwide. Recent biochemical and epidemiological studies underscore the importance of histo-blood group antigens (HBGA) as both cell attachment and susceptibility factors for the globally dominant P[4], P[6], and P[8] genotypes of human RVs. How these genotypes interact with HBGA is not known. Here, our crystal structures of P[4] and a neonate-specific P[6] VP8*s alone and in complex with H-type I HBGA reveal a unique glycan binding site that is conserved in the globally dominant genotypes and allows for the binding of ABH HBGAs, consistent with their prevalence. Remarkably, the VP8* of P[6] RVs isolated from neonates displays subtle structural changes in this binding site that may restrict its ability to bind branched glycans. This provides a structural basis for the age-restricted tropism of some P[6] RVs as developmentally regulated unbranched glycans are more abundant in the neonatal gut. Less
Angiotensin II AngII plays a central role in regulating human blood pressure which is mainly mediated by interactions between AngII and the G-protein-coupled receptors GPCRs AngII type receptor AT R and AngII type receptor AT R We have solved the crystal structure of human AT R binding the peptide ligand Sar Ile AngII and its specific antibody at - resolution Sar Ile AngII interacts with both the core binding domain where the small-molecule ligands of AT R and AT R bind and the extended binding domain which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor We ... More
Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R). We have solved the crystal structure of human AT2R binding the peptide ligand [Sar1, Ile8]AngII and its specific antibody at 3.2-Å resolution. [Sar1, Ile8]AngII interacts with both the ‘core’ binding domain, where the small-molecule ligands of AT1R and AT2R bind, and the ‘extended’ binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs. Less
The chemokine receptor CCR is an important anti-HIV human immunodeficiency virus drug target owning to its pivotal role in HIV- viral entry as a co-receptor Here we present a resolution crystal structure of CCR bound to PF- a second-generation oral CCR antagonist currently in phase II clinical trials PF- and the marketed HIV drug maraviroc share a similar tropane scaffold with different amino N - and carboxyl C - substituents Comparison of the CCR PF- structure with the previously determined structure of CCR in complex with maraviroc reveals different binding modes of the two allosteric antagonists and subsequent conformational changes ... More
The chemokine receptor CCR5 is an important anti-HIV (human immunodeficiency virus) drug target owning to its pivotal role in HIV-1 viral entry as a co-receptor. Here, we present a 2.9 Å resolution crystal structure of CCR5 bound to PF-232798, a second-generation oral CCR5 antagonist currently in phase II clinical trials. PF-232798 and the marketed HIV drug maraviroc share a similar tropane scaffold with different amino (N)- and carboxyl (C)- substituents. Comparison of the CCR5–PF-232798 structure with the previously determined structure of CCR5 in complex with maraviroc reveals different binding modes of the two allosteric antagonists and subsequent conformational changes of the receptor. Our results not only offer insights into the phenomenon that PF-232798 has higher affinity and alternative resistance profile to maraviroc, but also will facilitate the design of new anti-HIV drugs. Less
CCR is the primary chemokine receptor utilized by HIV to infect leukocytes whereas CCR ligands inhibit infection by blocking CCR engagement with HIV gp To guide the design of improved therapeutics we solved the structure of CCR in complex with chemokine antagonist P CCL Several structural features appeared to contribute to the anti-HIV potency of P CCL including the distinct chemokine orientation relative to the receptor the near-complete occupancy of the receptor binding pocket the dense network of intermolecular hydrogen bonds and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc Molecular modeling indicated that HIV gp mimicked ... More
CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokine interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases. Less
The Machine Recognition of Crystallization Outcomes MARCO initiative has assembled roughly half a million annotated images of macromolecular crystallization experiments from various sources and setups Here state-of-the-art machine learning algorithms are trained and tested on different parts of this data set We find that more than of the test images can be correctly labeled irrespective of their experimental origin Because crystal recognition is key to high-density screening and the systematic analysis of crystallization experiments this approach opens the door to both industrial and fundamental research applications
The bacterium Streptococcus pneumoniae the pneumococcus is a major human pathogen that requires Zn for its survival and virulence in the host environment Polyhistidine triad protein D PhtD has a known role in pneumococcal Zn homeostasis However the mechanistic basis of PhtD function remains unclear partly due to a lack of structural information Here we determined the crystal structure of the fragment PhtD - containing the third Zn -binding histidine triad HT motif of the protein Analysis of the structure suggests that Zn binding occurs at the surface of the protein and that all five HT motifs in the protein ... More
The bacterium Streptococcus pneumoniae (the pneumococcus) is a major human pathogen that requires Zn2+ for its survival and virulence in the host environment. Polyhistidine triad protein D (PhtD) has a known role in pneumococcal Zn2+ homeostasis. However, the mechanistic basis of PhtD function remains unclear, partly due to a lack of structural information. Here, we determined the crystal structure of the fragment PhtD269-339 , containing the third Zn2+ -binding histidine triad (HT) motif of the protein. Analysis of the structure suggests that Zn2+ binding occurs at the surface of the protein and that all five HT motifs in the protein bind Zn2+ and share similar structures. These new structural insights aid in our understanding of how the Pht proteins facilitate pneumococcal Zn2+ acquisition. Less
Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality However we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity which is critically required to enable rational design of degraders Here we utilize comprehensive characterization of the ligand dependent CRBN BRD interaction to demonstrate that binding between proteins that have not evolved to interact is plastic Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations which are selectively bound by ligands We demonstrate that computational protein-protein docking can ... More
Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize comprehensive characterization of the ligand dependent CRBN/BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations, which are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying inter-protein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic inter-protein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands. Less
Type-A -aminobutyric acid GABAA receptors are pentameric ligand-gated ion channels pLGICs typically consisting of subunit combinations They are the principal mediators of inhibitory neurotransmission throughout the central nervous system and targets of major clinical drugs such as benzodiazepines BZDs used to treat epilepsy insomnia anxiety panic disorder and muscle spasm However the structures of heteromeric receptors and the molecular basis of BZD operation remain unknown Here we report the cryo-EM structure of a human GABAAR in complex with GABA and a nanobody that acts as a novel positive allosteric modulator PAM The receptor subunits assume a unified quaternary activated conformation ... More
Type-A γ-aminobutyric acid (GABAA) receptors are pentameric ligand-gated ion channels (pLGICs), typically consisting of α/β/γ subunit combinations. They are the principal mediators of inhibitory neurotransmission throughout the central nervous system and targets of major clinical drugs, such as benzodiazepines (BZDs) used to treat epilepsy, insomnia, anxiety, panic disorder and muscle spasm. However, the structures of heteromeric receptors and the molecular basis of BZD operation remain unknown. Here we report the cryo-EM structure of a human α1β3γ2 GABAAR in complex with GABA and a nanobody that acts as a novel positive allosteric modulator (PAM). The receptor subunits assume a unified quaternary activated conformation around an open pore. We also present crystal structures of engineered α5 and α5γ2 GABAAR constructs, revealing the interfacial site for allosteric modulation by BZDs, including the binding modes and the conformational impact of the potent anxiolytic and partial PAM, bretazenil, and the BZD antagonist, flumazenil. These findings provide the foundation for understanding the mechanistic basis of GABAAR activation. Less
-Galactosidases EC are retaining glycosidases that cleave terminal -linked galactose residues from glycoconjugate substrates -Galactosidases take part in the turnover of cell wall associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals Deficiency of human -galactosidase A -Gal A causes Fabry disease FD a heritable X-linked lysosomal storage disorder characterized by accumulation of globotriaosylceramide Gb and globotriaosylsphingosine lyso-Gb Current management of FD involves enzyme-replacement therapy ERT An activity-based probe ABP covalently labeling the catalytic nucleophile of -Gal A has been previously designed to study -galactosidases for use in FD therapy Here we report that this ABP ... More
α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall–associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (Km = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management. Less
The process of producing suitable crystals for X-ray diffraction analysis most often involves the setting up of hundreds or thousands of individual crystallization trials each of which must be repeatedly examined for crystals or hints of crystallinity Currently the only real way to address this bottleneck is to use an automated imager to capture images of the trials However the images still need to be assessed for crystals or other outcomes Ideally there would exist some rapid and reliable machine-analysis tool to translate the images into a quantitative result However as yet no such tool exists in wide usage despite ... More
The process of producing suitable crystals for X-ray diffraction analysis most often involves the setting up of hundreds (or thousands) of individual crystallization trials, each of which must be repeatedly examined for crystals or hints of crystallinity. Currently, the only real way to address this bottleneck is to use an automated imager to capture images of the trials. However, the images still need to be assessed for crystals or other outcomes. Ideally, there would exist some rapid and reliable machine-analysis tool to translate the images into a quantitative result. However, as yet no such tool exists in wide usage, despite this being a well recognized problem. One of the issues in creating robust automatic image-analysis software is the lack of reliable data for training machine-learning algorithms. Here, a mobile application, Cinder, has been developed which allows crystallization images to be scored quickly on a smartphone or tablet. The Cinder scores are inserted into the appropriate table in a crystallization database and are immediately available to the user through a more sophisticated web interface, allowing more detailed analyses. A sharp increase in the number of scored images was observed after Cinder was released, which in turn provides more data for training machine-learning tools. Less
Platelet-activating-factor receptor PAFR responds to platelet-activating factor PAF a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects PAFR is considered an important drug target for treating asthma inflammation and cardiovascular diseases Here we report crystal structures of human PAFR in complex with the antagonist SR and the inverse agonist ABT- at - and - resolution respectively The structures supported by molecular docking of PAF provide insights into the signal-recognition mechanisms of PAFR The PAFR SR structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by and respectively and helix VIII ... More
Platelet-activating-factor receptor (PAFR) responds to platelet-activating factor (PAF), a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects. PAFR is considered an important drug target for treating asthma, inflammation and cardiovascular diseases. Here we report crystal structures of human PAFR in complex with the antagonist SR 27417 and the inverse agonist ABT-491 at 2.8-Å and 2.9-Å resolution, respectively. The structures, supported by molecular docking of PAF, provide insights into the signal-recognition mechanisms of PAFR. The PAFR–SR 27417 structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by 13 Å and 4 Å, respectively, and helix VIII adopts an inward conformation. The PAFR structures, combined with single-molecule FRET and cell-based functional assays, suggest that the conformational change in the helical bundle is ligand dependent and plays a critical role in PAFR activation, thus greatly extending knowledge about signaling by G-protein-coupled receptors. Less
The HECT E ligases ubiquitinate numerous transcription factors and signaling molecules and their activity must be tightly controlled to prevent cancer immune disorders and other diseases In this study we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities Biochemical structural and cellular analysis has revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications but complete removal of the brake can ... More
The HECT E3 ligases ubiquitinate numerous transcription factors and signaling molecules and their activity must be tightly controlled to prevent cancer, immune disorders, and other diseases. In this study we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities. Biochemical, structural, and cellular analysis has revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site. Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications, but complete removal of the brake can induce hyperactive autoubiquitination and E3 self-destruction. These results clarify the mechanisms of several HECT protein cancer associated mutations and provide a new framework for understanding how HECT ubiquitin ligases must be finely tuned to ensure normal cellular behavior. Less
MicroED structure of the NaK ion channel reveals a Na+ partition process into the selectivity filter
Sodium Na is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation signaling and facilitation of various transporters The hydration states of Na are proposed to play critical roles in determining the conductance and the selectivity of Na channels yet they are rarely captured by conventional structural biology means Here we use the emerging cryo-electron microscopy cryoEM method micro-electron diffraction MicroED to study the structure of a prototypical tetrameric Na -conducting channel NaK to resolution from nano-crystals Two new conformations at the external site of NaK are identified allowing us to visualize a partially ... More
Sodium (Na+) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na+ are proposed to play critical roles in determining the conductance and the selectivity of Na+ channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na+-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na+ ion at the entrance of the channel pore. A process of dilation coupled with Na+ movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics. Less
Protein phase diagrams are a tool to investigate the cause and consequence of solution conditions on protein phase behavior The effects are scored according to aggregation morphologies such as crystals or amorphous precipitates Solution conditions affect morphologic features such as crystal size as well as kinetic features such as crystal growth time Commonly used data visualization techniques include individual line graphs or phase diagrams based on symbols These techniques have limitations in terms of handling large data sets comprehensiveness or completeness To eliminate these limitations morphologic and kinetic features obtained from crystallization images generated with high throughput microbatch experiments have ... More
Protein phase diagrams are a tool to investigate the cause and consequence of solution conditions on protein phase behavior. The effects are scored according to aggregation morphologies such as crystals or amorphous precipitates. Solution conditions affect morphologic features, such as crystal size, as well as kinetic features, such as crystal growth time. Commonly used data visualization techniques include individual line graphs or phase diagrams based on symbols. These techniques have limitations in terms of handling large data sets, comprehensiveness or completeness. To eliminate these limitations, morphologic and kinetic features obtained from crystallization images generated with high throughput microbatch experiments have been visualized with radar charts in combination with the empirical phase diagram method. Morphologic features (crystal size, shape, and number, as well as precipitate size) and kinetic features (crystal and precipitate onset and growth time) are extracted for 768 solutions with varying chicken egg white lysozyme concentration, salt type, ionic strength, and pH. Image-based aggregation morphology and kinetic features were compiled into a single and easily interpretable figure, thereby showing that the empirical phase diagram method can support high-throughput crystallization experiments in its data amount as well as its data complexity. Less
Structure-based drug design SBDD heavily relies on the production of high-resolution three-dimensional D structures of the drug target in the presence or absence of the drug candidate X-ray crystallography is the predominant technique accounting for higher than of the structures in Protein Data Bank PDB considering the complexes involving protein and inhibitors Macromolecular X-ray crystallography provides an important and powerful technique in studying the specific interactions of a particular drug with its protein target at the atomic level that can help improve the drug design process It is the main technique used to obtain D information for binary complexes involving ... More
Structure-based drug design (SBDD) heavily relies on the production of high-resolution (<2Å) three-dimensional (3D) structures of the drug target in the presence or absence of the drug candidate [1]. X-ray crystallography is the predominant technique accounting for higher than 94% of the structures in Protein Data Bank (PDB), considering the complexes involving protein and inhibitors [2–4]. Macromolecular X-ray crystallography provides an important and powerful technique in studying the specific interactions of a particular drug with its protein target at the atomic level that can help improve the drug design process [5]. It is the main technique used to obtain 3D information for binary complexes involving protein and drugs [6]. One of the major ‘bottlenecks’ in X-ray crystallography is the lack of generalized methods for high quality crystal production. Since protein crystallization mechanism details remain unknown, protein crystallization is a complicated and time-consuming process and requires performing a significant number of trial-and-error experiments involving systematic testing of variable chemical and physical parameters [7]. High-throughput (HT) structural biology coincided with the dawn of the genomic era in biology requires the automation, miniaturization, and parallelization of protein crystallization in order to reach the capacity necessary for large-scale structure determination efforts [8]. HT protein crystallization screening (HTPCS) technologies appeared on the protein crystallization scene more than three decades ago and have since allowed accessing hundreds to thousands of protein crystallization conditions, thereby greatly impacting HT structural biology [9]. HTPCS has helped to identify critical components required for HT crystallization efforts [7]. In recent years, the rapid developments of manipulation techniques and devices provide effective and reliable solutions for protein crystallization screening with HT and low consumption. In spite of their advances, HTPCS has suffered from two main handicaps, namely, poor hit rate in protein crystallization screening and lack of predictive power of the scoring functions. To overcome these handicaps, several projects have recently been initiated to construct ‘smart systems’ that are not only capable of rapidly performing a large number of crystallization trials, but also scripting and triggering certain events based on the collected data used in predict the outcome of a protein x-ray crystallization trial [10]. In this contest, we will outline recent efforts in HTPCS that could improve the success rate of the structural pipeline. We will discuss the challenge and some of the possible avenues in that direction. Less
Neuropeptide Y NPY receptors belong to the G protein-coupled receptor GPCR superfamily and play important roles in food intake anxiety and cancer regulation The NPY Y receptor system has emerged as one of the most complex networks with three peptide ligands NPY peptide YY and pancreatic polypeptide binding to four receptors in mammals namely Y Y Y and Y receptors with different affinity and selectivity NPY is the most powerful stimulant of food intake and this effect is primarily mediated by Y receptor Y R A number of peptides and small-molecule compounds have been characterized as Y R antagonists and ... More
Neuropeptide Y (NPY) receptors belong to the G protein-coupled receptor (GPCR) superfamily and play important roles in food intake, anxiety and cancer regulation1,2. The NPY/Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in mammals, namely Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity3. NPY is the most powerful stimulant of food intake and this effect is primarily mediated by Y1 receptor (Y1R)4. A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity4, tumor1 and bone loss5. However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability6. Here we report crystal structures of the human Y1R bound to two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal binding modes of Y1R to several structurally diverse antagonists and determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance (NMR), photo-crosslinking and functional studies, provide insights into the binding behavior of the agonist and for the first time determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery targeting NPY receptors. Less
Epstein-Barr virus EBV is a causative agent of infectious mononucleosis and is associated with new cases of cancer and deaths annually Subunit vaccines against this pathogen have focused on the gp glycoprotein and remain unsuccessful We isolated human antibodies recognizing the EBV fusion machinery gH gL and gB from rare memory B cells One anti-gH gL antibody AMMO potently neutralized infection of B cells and epithelial cells the two major cell types targeted by EBV We determined a cryo-electron microscopy reconstruction of the gH gL-gp -AMMO complex and demonstrated that AMMO bound to a discontinuous epitope formed by both gH ... More
Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and is associated with 200,000 new cases of cancer and 140,000 deaths annually. Subunit vaccines against this pathogen have focused on the gp350 glycoprotein and remain unsuccessful. We isolated human antibodies recognizing the EBV fusion machinery (gH/gL and gB) from rare memory B cells. One anti-gH/gL antibody, AMMO1, potently neutralized infection of B cells and epithelial cells; the two major cell types targeted by EBV. We determined a cryo-electron microscopy reconstruction of the gH/gL-gp42-AMMO1 complex and demonstrated that AMMO1 bound to a discontinuous epitope formed by both gH and gL at the Domain-I/Domain-II interface. Integrating structural, biochemical and infectivity data, we propose that AMMO1 inhibits fusion of the viral and cellular membranes. This work identifies a crucial epitope that may aid in the design of next-generation subunit vaccines against this major public health burden. Less
The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein related lipid transfer StART domains that are implicated in intracellular non-vesicular lipid transport A new family of membrane-anchored StARkins was recently identified including six members Lam Lam in the yeast Saccharomyces cerevisiae Lam Lam are anchored to the endoplasmic reticulum ER membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast To better understand the biological roles of these proteins we carried out a structure-function analysis of the second StARkin domain of Lam here termed Lam S NMR ... More
The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein–related lipid transfer (StART) domains that are implicated in intracellular, non-vesicular lipid transport. A new family of membrane-anchored StARkins was recently identified, including six members, Lam1–Lam6, in the yeast Saccharomyces cerevisiae. Lam1–Lam4 are anchored to the endoplasmic reticulum (ER) membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast. To better understand the biological roles of these proteins, we carried out a structure-function analysis of the second StARkin domain of Lam4, here termed Lam4S2. NMR experiments indicated that Lam4S2 undergoes specific conformational changes upon binding sterol, and fluorescence-based assays revealed that it catalyzes sterol transport between vesicle populations in vitro, exhibiting a preference for vesicles containing anionic lipids. Using such vesicles, we found that sterols are transported at a rate of ∼50 molecules per Lam4S2 per minute. Crystal structures of Lam4S2, with and without bound sterol, revealed a largely hydrophobic but surprisingly accessible sterol-binding pocket with the 3-OH group of the sterol oriented toward its base. Single or multiple alanine or aspartic acid replacements of conserved lysine residues in a basic patch on the surface of Lam4S2 near the likely sterol entry/egress site strongly attenuated sterol transport. Our results suggest that Lam4S2 engages anionic membranes via a basic surface patch, enabling “head-first” entry of sterol into the binding pocket followed by partial closure of the entryway. Reversal of these steps enables sterol egress. Less
Mitochondrial Carriers MCs are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism The ADP ATP Carriers AACs are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP In human four different AACs isoforms are described which are expressed in tissue-specific manner They are involved in different genetic diseases and play a role in cancerogenesis Up to now only the structures of the bovine isoform and yeast isoforms and AAC have been determined in one particular conformation obtained in complex ... More
Mitochondrial Carriers (MCs) are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism. The ADP/ATP Carriers (AACs) are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP. In human, four different AACs isoforms are described which are expressed in tissue-specific manner. They are involved in different genetic diseases and play a role in cancerogenesis. Up to now only the structures of the bovine (isoform 1) and yeast (isoforms 2 and 3) AAC have been determined in one particular conformation, obtained in complex with the CATR inhibitor. Herein, we report that full-length human ADP/ATP Carriers isoform 1 and 3 were successfully expressed in cell-free system and purified in milligram amounts in detergent-solubilized state. The proteins exhibited the expected secondary structure content. Thermostability profiles showing stabilization by the CATR inhibitor suggest that the carriers are well folded. Less
Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum Pf circumsporozoite protein PfCSP from several subjects immunized with an attenuated Pf whole-sporozoite SPZ vaccine Sanaria PfSPZ Vaccine Passive transfer of one of these antibodies monoclonal antibody CIS conferred high-level sterile protection in two different mouse models of malaria infection The affinity and stoichiometry of CIS binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain The first ... More
Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed. Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) from several subjects immunized with an attenuated Pf whole-sporozoite (SPZ) vaccine (Sanaria PfSPZ Vaccine). Passive transfer of one of these antibodies, monoclonal antibody CIS43, conferred high-level, sterile protection in two different mouse models of malaria infection. The affinity and stoichiometry of CIS43 binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain. The first binding event is to a unique 'junctional' epitope positioned between the N terminus and the central repeat domain of PfCSP. Moreover, CIS43 prevented proteolytic cleavage of PfCSP on PfSPZ. Analysis of crystal structures of the CIS43 antigen-binding fragment in complex with the junctional epitope determined the molecular interactions of binding, revealed the epitope's conformational flexibility and defined Asn-Pro-Asn (NPN) as the structural repeat motif. The demonstration that CIS43 is highly effective for passive prevention of malaria has potential application for use in travelers, military personnel and elimination campaigns and identifies a new and conserved site of vulnerability on PfCSP for next-generation rational vaccine design. Less
As a protective envelope surrounding the bacterial cell the peptidoglycan sacculus is a site of vulnerability and an antibiotic target Peptidoglycan components assembled in the cytoplasm are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate A product of peptidoglycan synthesis undecaprenyl-pyrophosphate is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase BacA To understand how BacA functions we determine its crystal structure at resolution The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane Conserved residues map to the pocket where pyrophosphorolysis occurs BacA ... More
As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase. Less
Investigation of the biochemical basis of life is a long standing challenge for scientists The field of molecular biology was established based on the pioneering studies of macromolecular structures including the structural characterization of the DNA double helix and the description of the first protein structures These early achievements along with the technical development e g automatic data collection and computer-assisted data analysis enables extremely efficient and fast determination of structure and dynamic properties of proteins
The low limits of detection afforded by second harmonic generation SHG microscopy coupled with image analysis algorithms enabled quantitative modeling of the temperature-dependent crystallization of active pharmaceutical ingredients APIs within amorphous solid dispersions ASDs ASDs in which an API is maintained in an amorphous state within a polymer matrix are finding increasing use to address solubility limitations of small-molecule APIs Extensive stability testing is typically performed for ASD characterization the time frame for which is often dictated by the earliest detectable onset of crystal formation Here a study of accelerated stability testing on ritonavir a human immunodeficiency virus HIV protease ... More
The low limits of detection afforded by second harmonic generation (SHG) microscopy coupled with image analysis algorithms enabled quantitative modeling of the temperature-dependent crystallization of active pharmaceutical ingredients (APIs) within amorphous solid dispersions (ASDs). ASDs, in which an API is maintained in an amorphous state within a polymer matrix, are finding increasing use to address solubility limitations of small-molecule APIs. Extensive stability testing is typically performed for ASD characterization, the time frame for which is often dictated by the earliest detectable onset of crystal formation. Here a study of accelerated stability testing on ritonavir, a human immunodeficiency virus (HIV) protease inhibitor, has been conducted. Under the condition for accelerated stability testing at 50 �C/75%RH and 40 �C/75%RH, ritonavir crystallization kinetics from amorphous solid dispersions were monitored by SHG microscopy. SHG microscopy coupled by image analysis yielded limits of detection for ritonavir crystals as low as 10 ppm, which is about 2 orders of magnitude lower than other methods currently available for crystallinity detection in ASDs. The four decade dynamic range of SHG microscopy enabled quantitative modeling with an established (JMAK) kinetic model. From the SHG images, nucleation and crystal growth rates were independently determined. Less
Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the -oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA RNA production This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring To provide validation for this approach as a drug target we have RNAi silenced the three -oxopurine phosphoribosyltransferase PRTase isoforms in the infectious stage of Trypanosoma brucei demonstrating that ... More
Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites’ viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery. Less
Plastidial thioredoxin TRX -like proteins are atypical thioredoxins possessing a WCRKC active site signature and using glutathione for recycling To obtain structural information supporting the peculiar catalytic mechanisms and target proteins of these TRXs we solved the crystal structures of poplar TRX-like in oxidized and reduced states and of mutated variants These structures share similar folding with TRXs exhibiting the canonical WCGPC signature Moreover the overall conformation is not altered by reduction of the catalytic disulfide bond or in a C S C S variant that formed a disulfide-bridged dimer possibly mimicking reaction intermediates with target proteins Modeling of the ... More
Plastidial thioredoxin (TRX)-like2.1 proteins are atypical thioredoxins possessing a WCRKC active site signature and using glutathione for recycling. To obtain structural information supporting the peculiar catalytic mechanisms and target proteins of these TRXs, we solved the crystal structures of poplar TRX-like2.1 in oxidized and reduced states and of mutated variants. These structures share similar folding with TRXs exhibiting the canonical WCGPC signature. Moreover, the overall conformation is not altered by reduction of the catalytic disulfide bond or in a C45S/C67S variant that formed a disulfide-bridged dimer possibly mimicking reaction intermediates with target proteins. Modeling of the interaction of TRX-like2.1 with both NADPH- and ferredoxin-thioredoxin reductases (FTR) indicates that the presence of Arg43 and Lys44 residues likely precludes reduction by the plastidial FTR. Less
In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains ECDs comparable to the water channels nm of the mesophase Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid Ia d double-diamond Pn m and double-primitive Im m space groups with water channels five times larger than traditional lipidic mesophases and showing re-entrant behavior upon increasing hydration of sequences Ia d ... More
In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3�5 nm) of the mesophase. Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d?Pn3m?Ia3d and Pn3m?Im3m?Pn3m, unknown in lipid self-assembly. We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology. Less
Drugs frequently require interactions with multiple targets via a process known as polypharmacology to achieve their therapeutic actions Currently drugs targeting several serotonin receptors including the -HT C receptor are useful for treating obesity drug abuse and schizophrenia The competing challenges of developing selective -HT C receptor ligands or creating drugs with a defined polypharmacological profile especially aimed at G protein-coupled receptors GPCRs remain extremely difficult Here we solved two structures of the -HT C receptor in complex with the highly promiscuous agonist ergotamine and the -HT A-C receptor-selective inverse agonist ritanserin at resolutions of and respectively We analyzed their ... More
Drugs frequently require interactions with multiple targets — via a process known as polypharmacology — to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin, at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs. Less
The ageing suppressor -klotho binds to the fibroblast growth factor receptor FGFR This commits FGFR to respond to FGF a key hormone in the regulation of mineral ion and vitamin D homeostasis The role and mechanism of this co-receptor are unknown Here we present the atomic structure of a ternary complex that consists of the shed extracellular domain of -klotho the FGFR c ligand-binding domain and FGF In this complex -klotho simultaneously tethers FGFR c by its D domain and FGF by its C-terminal tail thus implementing FGF FGFR c proximity and conferring stability Dimerization of the stabilized ternary complexes ... More
The ageing suppressor α-klotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion and vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex that consists of the shed extracellular domain of α-klotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, α-klotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23–FGFR1c proximity and conferring stability. Dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signalling. The structure of α-klotho is incompatible with its purported glycosidase activity. Thus, shed α-klotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signalling. Less
The maturation of Ras GTPases and other cellular CaaX proteins involves three enzymatic steps addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine C in the C-terminal CaaX motif proteolytic cleavage of the aaX residues and methylation of the exposed prenylcysteine residue at its terminal carboxylate This final step is catalyzed by isoprenylcysteine carboxyl methyltransferase ICMT a eukaryotic-specific integral membrane enzyme of the endoplasmic reticulum ER ICMT is the only cellular enzyme known to methylate prenylcysteine substrates methylation is important for their biological functions including the membrane localisations and subsequent activities of Ras prelamin A and Rab ICMT ... More
The maturation of Ras GTPases, and ~200 other cellular CaaX proteins, involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CaaX motif, proteolytic cleavage of the aaX residues, and methylation of the exposed prenylcysteine residue at its terminal carboxylate1. This final step is catalyzed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryotic-specific integral membrane enzyme of the endoplasmic reticulum (ER)2. ICMT is the only cellular enzyme known to methylate prenylcysteine substrates; methylation is important for their biological functions, including the membrane localisations and subsequent activities of Ras1, prelamin A3, and Rab4. ICMT inhibition has potential for combating progeria3 and cancer5–8. Here we present an X-ray structure of ICMT, at 2.3 Å resolution, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for its cytosolic methyl donor, S-adenosyl-L-methionine (AdoMet), and for prenylcysteine substrates, which are associated with the ER membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localisations together in a membrane environment – a relatively uncharacterized, but defining feature of many integral membrane enzymes. Less
Canonical FGFs activate FGF receptors FGFR via paracrine or autocrine mechanisms in a process requiring cooperation with heparan sulfate proteoglycans that function as co-receptors for FGFR activation By contrast endocrine FGFs FGF and are circulating hormones that regulate critical metabolic processes in a variety of tissues FGF regulates bile acid synthesis and lipogenesis while FGF stimulate insulin sensitivity energy expenditure and weight loss Endocrine FGFs signal through FGFRs in a manner that requires Klothos which are cell surface proteins with tandem glycosidase domains Here we describe the crystal structures of free and ligand-bound -Klotho extracellular regions revealing the molecular mechanism ... More
Canonical FGFs activate FGF receptors (FGFR) via paracrine or autocrine mechanisms, in a process requiring cooperation with heparan sulfate proteoglycans that function as co-receptors for FGFR activation1,2. By contrast, endocrine FGFs (FGF 19, 21, and 23) are circulating hormones that regulate critical metabolic processes in a variety of tissues3,4. FGF19 regulates bile acid synthesis and lipogenesis, while FGF21 stimulate insulin sensitivity, energy expenditure and weight loss5. Endocrine FGFs signal through FGFRs in a manner that requires Klothos, which are cell surface proteins with tandem glycosidase domains3,4. Here we describe the crystal structures of free and ligand-bound β-Klotho extracellular regions, revealing the molecular mechanism underlying the specificity of FGF21 towards β-Klotho and demonstrating how FGFR is activated in a Klotho-dependent manner. β-Klotho serves as a primary “Zip code”-like receptor for FGF21 with an FGFR functioning as a catalytic subunit that mediates intracellular signaling. Our structures also show how a sugar cutting enzyme (glycosidase) has evolved to become a specific receptor for hormones that regulate metabolic processes including lowering of blood sugar. Finally, we describe a superior agonistic variant of FGF21 and present structural insights offering development of novel therapeutics for diseases linked to endocrine FGFs. Less